The zebrafish model system has become a main model used to dissect gene function and molecular mechanisms during vertebrate development disease. Forward genetic strategies have been particularly powerful and have identified many genes involved in development and disease. However, for a variety of reasons, some critical genes and/or pathways will not be identified in forward screens, necessitating the need for reverse genetic strategies. Of particular interest to the zebrafish research community is the development of gene targeting strategies that will allow the precise modification of a gene locus, e.g., to create an allele mimicking a human disease mutation, tag the endogeneous locus with a marker protein, and/or to generate conditional alleles. A promising new reverse genetics approach is the use of zinc finger nucleases (ZFNs) to induce double strand breaks (DSBs) at precise positions within zebrafish loci. Induced DSBs can be repaired via non-homologous end joining (NHEJ), which is often mutagenic, creating small insertions and deletions at the DSB site. Alternatively, DSBs can be repaired by homology directed repair (HDR), using either the homologous chromosome or an exogenously supplied donor containing significant homology. We have shown that in zebrafish, NHEJ-mediated repair of ZFN-induced DSBs occurs readily and efficiently in both somatic cells and the germline. In our experiments, we show that over 60% of zebrafish embryos injected with ZFN-encoding mRNA and grow to adulthood carry new alleles at germline frequencies averaging 20%. We propose here to optimize the use of ZFNs for HDR and gene targeting. Specifically, we propose to optimize conditions for ZFN-induced gene targeting in zebrafish, to characterize whether ZFN-mediated HDR can be enhanced by manipulating the developmental timing at which the DSBs are induced or by manipulating components that regulate DSB repair pathway choice, and to distribute detailed protocols and reagents to the zebrafish community. Development of protocols and tools that facilitate gene targeting in zebrafish will have a huge impact on the field. The ability to engineer precise sequence changes, instead of relying on random mutagenesis, will allow researchers to generate the types of alleles that are critically relevant to human health and disease, including conditional null alleles, which allow one to study gene function in specific tissues or at specific times that is normally masked due to pleiotropic function, and models of human disease by engineering specific human mutations into the gene of interest. PUBLIC HEALTH RELEVANCE: Forward and reverse genetic strategies in vertebrate model organisms, such as the zebrafish, and the subsequent phenotypic analysis of mutant phenotypes, has provided critical insight into genes, pathways, and mechanisms regulating human development and disease. Zebrafish forward genetic strategies have been important for new gene discovery, and more recently, zebrafish reverse genetic strategies have allowed recovery of mutations in specific genes of interest. That said, targeted genome manipulation in vertebrates, the precise and specific modification of gene sequence, has been largely restricted to the mouse, due to the availability of mouse embryonic stem cell lines that can be manipulated in culture and reintroduced into mouse blastocysts. With the discovery that designed zinc finger nucleases (ZFNs) can be used to generate double-strand breaks at precise positions in the zebrafish genome, we are now poised to optimize this technology for gene targeting, facilitating the construction of conditional alleles, tagged alleles, or disease models carrying human disease allele mutations, among many other exciting possibilities. Such strategies will become critically important as we begin to dissect signaling pathways that control developmental processes at multiple times and in multiple tissues and to model human diseases with later-onset phenotypes, as wide-ranging as behavioral disorders, neuromuscular disease, and cancer. We propose here to develop protocols and tools for optimization of ZFN-mediated gene targeting and rapidly disseminate them to the zebrafish community.